To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.

中文翻译：

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热荧光适体结合测定的特质。

为了详细探讨热荧光分析（TFA），我们比较了两种相关的适体。第一种，LINN2，是先前针对EGFR重组蛋白选择的DNA适体。在这项工作中，我们选择了另一种针对EGFR过表达的A549细胞的适体KM4。这两个适体源自相同的池并结合相同的靶标，但在TFA中表现不同。我们的结果提出了有关适体TFA的四个总体结论：1.一些适体在靶标结合后显示出降低的荧光，这表明与靶标结合的适体并不总是发荧光的。2.许多适体不遵循直觉假设，即适体-靶标相互作用稳定折叠构象。3.TFA对于具有明显双链结构的适体可能是最合适的。4。